Optical sensor of bio-molecules using thin-film interferometer

ABSTRACT

The present invention is directed to an assembly for use in detecting an analyte in a sample based on thin-film spectral interference. The assembly comprises a waveguide, a monolithic substrate optically coupled to the waveguide, and a thin-film layer directly bonded to the sensing side of the monolithic substrate. The refractive index of the monolithic substrate is higher than the refractive index of the transparent material of the thin-film layer. A spectral interference between the light reflected into the waveguide from a first reflecting surface and a second reflecting surface varies as analyte molecules in a sample bind to the analyte binding molecules coated on the thin-film layer.

This application is a continuation of PCT/US2010/024801, filed Feb. 19,2010; which claims the priority of U.S. Provisional Application Nos.61/208,215, filed Feb. 20, 2009; and 61/279,077, filed Oct. 15, 2009.The contents of the above-identified applications are incorporatedherein by reference in their entirety.

TECHNICAL FIELD

This invention relates to an apparatus that improves detecting thepresence, amount, or rate of binding of one or more analytes in asample, and in particular an apparatus utilizing thin-filminterferometer technology.

BACKGROUND OF THE INVENTION

Diagnostic tests based on a binding event between members of ananalyte-anti-analyte binding pair are widely used in medical,veterinary, agricultural and research applications. Typically, suchmethods are employed to detect the presence or amount or an analyte in asample, and/or the rate of binding of the analyte to the anti-analyte.Typical analyte-anti-analyte pairs include complementary strands ofnucleic acids, antigen-antibody pairs, and receptor-receptor bindingagent, where the analyte can be either member of the pair, and theanti-analyte molecule, the opposite member.

Diagnostics methods of this type often employ a solid surface havingimmobilized anti-analyte molecules to which sample analyte moleculeswill bind specifically and with high affinity at a defined detectionzone. In this type of assay, known as a solid-phase assay, the solidsurface is exposed to the sample under conditions that promote analytebinding to immobilized anti-analyte molecules. The binding event can bedetected directly, e.g., by a change in the mass, reflectivity,thickness, color or other characteristic indicative of a binding event.Where the analyte is pre-labeled, e.g., with a chromophore, orfluorescent or radiolabel, the binding event is detectable by thepresence and/or amount of detectable label at the detection zone.Alternatively, the analyte can be labeled after it is bound at thedetection zone, e.g., with a secondary, fluorescent-labeled anti-analyteantibody.

U.S. Pat. No. 5,804,453 discloses a method of determining theconcentration of a substance in a sample solution, using a fiber optichaving a reagent (capturing molecule) coated directly at its distal endto which the substance binds. The distal end is then immersed into thesample containing the analyte. Binding of the analyte to the reagentlayer generates an interference pattern and is detected by aspectrometer.

U.S. Pat. No. 7,394,547 discloses a biosensor that a first opticallytransparent element is mechanical attached to an optic fiber tip with anair gap between them, and a second optical element as the interferencelayer with a thickness greater than 50 nm is then attached to the distalend of the first element. The biolayer is formed on the peripheralsurface of the second optical element. An additional reflective surfacelayer with a thickness between 5-50 nm and a refractive index greaterthan 1.8 is coated between the interference layer and the first element.The principle of detecting an analyte in a sample based on the changesof spectral interference is described in this reference, which isincorporated herein by reference.

U.S. Pat. No. 7,319,525 discloses a different configuration that asection of an optic fiber is mechanically attached to a tip connectorconsisting of one or more optic fibers with an air gap between theproximal end of the optic fiber section and the tip connector. Theinterference layer and then the biolayer are built on the distal surfaceof the optical fiber section.

An air gap between coupling fibers has several disadvantages. Onedrawback is the reduction in coupling efficiency. Another problem ispoor alignment. Practically, it is difficult to maintain the same exactair gap size for different pairs of sensors and instruments. Inaddition, air-fiber interface tends to causing higher reflection back tothe spectrometer that can decrease the measurement signal-to-noiseratio.

Although prior art provides functionality in utilizing bio-sensors basedupon thin-film interferometer, there exists a need for improvements inthe performance of the interferometer.

SUMMARY OF THE INVENTION

The present invention is directed to an assembly for use in detecting ananalyte in a sample based on thin-film spectral interference. Theassembly comprises a waveguide connector containing a waveguide, whereinthe waveguide transports a light signal from a light source to theassembly, and the waveguide transports reflected light signals from theassembly to a detector. The assembly further comprises a monolithicsubstrate having a coupling side and a sensing side, the coupling sideis coupled to the waveguide connector by a coupling hub, wherein thewaveguide connector is engaged with the coupling hub to form a opticalcoupling between the waveguide and the monolithic substrate. Theassembly further comprises a coupling medium located between thewaveguide connector and the monolithic substrate so the end of thewaveguide couples to the monolithic substrate without any gap. Theassembly additionally comprises a thin-film layer directly bonded to thesensing side of the monolithic substrate, wherein the thin film layercomprises a transparent material, a first reflecting surface comprisinga layer of analyte binding molecules, and a second reflecting surfacebetween the thin film layer and the monolithic substrate. A spectralinterference between light reflected into the waveguide from theassembly varies as analyte molecules in the sample bind to the analytebinding molecules.

In one embodiment, the coupling hub is inserted into the waveguideconnector. In another embodiment, the waveguide connector is a ferruleand the ferrule is inserted into the coupling hub.

The present invention is also directed to an assembly comprising: (a) awaveguide; (b) a monolithic substrate having a coupling side and asensing side, the coupling side is optically coupled to the waveguide;and (d) a thin-film layer directly bonded to the sensing side of themonolithic substrate. The waveguide transports a light signal from alight source to the assembly, and the waveguide transports reflectedlight signals from the assembly to a detector. The thin film layercomprises a transparent material, a first reflecting surface comprisinga layer of analyte binding molecules, and a second reflecting surfacebetween the thin film layer and the monolithic substrate. The refractiveindex of the monolithic substrate is higher than the refractive index ofthe transparent material of the thin-film layer; and the refractiveindex of the coupling medium is greater than 1.3, preferably in betweenrefractive indexes of the waveguide and the monolithic substrate. Aspectral interference between light reflected into the waveguide fromsaid first and said second reflecting surfaces varies as analytemolecules in the sample bind to the analyte binding molecules. In oneembodiment, the assembly further comprises a conpling medium between thewaveguide and the monolithic substrate, wherein the refractive index ofthe coupling medium is greater than 1.3.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the general embodiment of the present invention.

FIG. 2A and 2B illustrate the principles of detection in a thin-filminterferometer.

FIG. 3 illustrates a probe inserted into a coupling hub.

FIG. 4 illustrates a biosensor with a coupling hub.

FIG. 5 illustrates the present invention implemented with a couplinghub.

FIG. 6A-C illustrates one coupling hub embodiment with coupling mediumpre-installed inside the hub.

FIG. 7A-E illustrates a second coupling hub embodiment with a drop ofcoupling medium formed on the tip of the ferrule.

FIG. 8A-C illustrates a third coupling hub embodiment with a couplingmedium attached to the ferrule.

FIG. 9A-B illustrates a fourth coupling hub embodiment with the couplinghub comprising a coupling medium.

FIG. 10A-C illustrates another coupling hub embodiment with the couplinghub comprising a coupling medium.

FIG. 11A-C illustrates another coupling hub embodiment with the couplingmedium attached to the waveguide connector.

FIG. 12 illustrates a Y coupler with uneven branches.

FIG. 13 illustrates a typical interference pattern of a binding assaydetected by a thin-film interferometer.

FIG. 14 illustrates the association and dissociation curves of twoprobes simultaneously.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms used in the claims and specification are to be construed inaccordance with their usual meaning as understood by one skilled in theart except and as defined as set forth below.

“About,” as used herein, refers to within ±15% of the recited value.

An “analyte-binding” molecule, as used herein, refers to any moleculecapable of participating in a specific binding reaction with an analytemolecule. Examples include but are not limited to, (i) antigenmolecules, for use in detecting the presence of antibodies specificagainst that antigen; (ii) antibody molecules, for use in detecting thepresence of antigens; (iii) protein molecules, for use in detecting thepresence of a binding partner for that protein; (iv) ligands, for use indetecting the presence of a binding partner; or (v) single strandednucleic acid molecules, for detecting the presence of nucleic acidbinding molecules.

A “ferrule” as used herein, refers to a rigid tube that confines orholds a waveguide as part of a connector assembly.

“A monolithic substrate,” as used herein, refers to a single piece of asolid material such as glass, quartz, or plastic that has one refractiveindex.

A “probe,” as used herein, refers to a monolithic substrate coated witha thin-film layer at the sensing side.

A “waveguide” as used herein, refers to a device (as a duct, coaxialcable, or optic fiber) designed to confine and direct the propagation ofelectromagnetic waves (as light); for example, a waveguide is a metaltube for channeling ultrahigh-frequency waves.

A “waveguide connector” as used herein, refers to a mechanical devicefor optically joining the locking together separable mating parts of awaveguide system. It is also known as a waveguide coupler.

The inventors have discovered that using a coupling medium instead of anair gap to couple between a monolithic substrate and a waveguide canreduce the coupling loss and decreases the reflections from the surfaceof the waveguide and the surface of the monolithic substrate. With thecoupling medium, the coupling efficiency is greatly improved, theundesirable reflection to the spectrometer is reduced, and the alignmentproblem due to an air gap is revolved.

The inventors also discovered the use of a monolithic substrate in anoptical assembly with higher refractive index over the interferencelayer, hence eliminating the need for coating an extra layer of a highrefractive index material on the interference layer, between theinterference layer and the monolithic substrate.

FIG. 1 illustrates a general embodiment of the present invention: abio-sensor interferometer 10 comprising a light source 11, a detector12, waveguide 13 and an optical assembly 14. The optical assembly 14comprises the tip of the waveguide 15, a coupling medium 16, amonolithic substrate 17, a thin film layer (interference layer) 22 and abiomolecular layer 21. The thin film layer comprises a transparentmaterial, a first reflecting surface comprising a layer of biomolecularmolecules 21, and a second reflecting surface 23 between the thin filmlayer and the monolithic substrate. Also at the interface between thethin film layer 22 and the biomolecular layer 21 is a sensing surface24.

A light source 11 in the apparatus can be a white light source, such asa light emitting diode (LED) which produces light over a broad spectrum,e.g., 400 nm or less to 700 nm or greater, typically over a spectralrange of at least 100 nm. Alternatively, a light source can be aplurality of sources each having a different characteristic wavelength,such as LEDs designed for light emission at different selectedwavelengths in the visible light range. The same function can beachieved by a single light source, e.g., white light source, withsuitable filters for directing light with different selected wavelengthsonto the optical assembly.

A detector 12 is preferably a spectrometer, such as Ocean OpticsUSB4000, capable of recording the spectrum of the reflected interferinglight from the optical assembly. Alternatively, where the light sourceoperates to direct different selected wavelengths onto the opticalassembly, the detector can be a simple photodetector for recording lightintensity at each of the different irradiating wavelengths. In stillanother embodiment, the detector can include a plurality of filterswhich allows detection of light intensity, e.g., from a white-lightsource, at each of a plurality of selected wavelengths of theinterference reflectance wave.

A waveguide 13 transports a light signal from a light source to anoptical assembly, and transports reflected light signals from theoptical assembly to a detector. Commonly the waveguide comprises a fiberbundle. Spectrometer is a typical light detector used in bio-sensorinterferometers.

A coupling medium 16 couples the waveguide and the monolithic substratewithout leaving any gap between them. The coupling medium is anoptically transparent material such as a polymer, an index matching gel,or an index matching liquid with a desired specific refractive index. Anindex matching liquid should have desired physical and chemicalproperties such as high viscosity, low vapor pressure (not easilyevaporated) and a low corrosion rate. Suitable materials for thecoupling medium comprise the following: polycarbonate, poly(methylmethacrylate) (PMMA), polystyrene (PS), polypropylene (PP),acrylonitrile butadiene styrene (ABS), refractive index matching gel,refractive index matching liquid, or polydimethylsiloxane (PDMS).Typically, the waveguide 13 comprises fiber material having a refractiveindex of approximately 1.5. Since the refractive index of the air gap isapproximately 1.00, the presence of an air gap between the waveguide andthe monolithic substrate would result in a coupling loss and highreflection. When the coupling medium couples the waveguide and themonolithic substrate without leaving any gap between them, the couplingefficiency is greatly improved, reflection is reduced, and alignmentproblems are alleviated. In a preferred embodiment, the refractive indexof the coupling medium is in between the refractive indexes of thewaveguide and the monolithic substrate.

A monolithic substrate made of a monolithic dielectric material iscoupled to a tip of a waveguide through a coupling medium. The crosssection of the monolithic substrate may be round, square, triangular,oval, or rectangular shaped. In a preferred embodiment, the aspect ratioof the monolithic substrate (length to width or length to diameter) isat least 5:1. The monolithic substrate material preferably has arefractive index that is substantially higher than that of the thin-filmlayer, such that the second reflective surface effectively reflects aportion of the light directed onto the optical assembly. The preferredrefractive index of the monolithic substrate material is higher than1.5, or higher than 1.8, or higher than 2.0. A preferred refractiveindex range of the monolithic substrate material is between about 1.55to about 2.0. The monolithic substrate works effectively as an opticalwaveguide when the incident light enters the substrate proximal surfacein a certain angle that still allows a total internal reflection. Inthis embodiment, this angle is defined by the combination of thenumerical aperture of the lighting optic fiber, the substrate, and themechanical coupling angel between the substrate and the lighting opticfiber.

An interference layer (a thin-film layer) is a transparent materialcoated on the sensing side of the monolithic substrate. Thin films arethin material layers ranging from fractions of a nanometer (monolayer)to several micrometers in thickness. Electronic semiconductor devicesand optical coatings are the main applications benefiting from thin filmconstruction. The thin-film layer of the present invention typically hasa thickness of at least 50 nm, and preferably at least 100 nm. Anexemplary thickness is between about 100-5,000 nm, preferably 400-1,000nm. The refractive index of the thin-film layer material is preferablysimilar to that of the first reflecting surface, so that reflection fromthe lower distal end of the optical assembly occurs predominantly fromthe layer formed by the analyte-binding molecules, rather than from theinterface between the optical element and the analyte-binding molecules.Similarly, as analyte molecules bind to the lower layer of the opticalassembly, light reflection form the lower end of the assembly occurspredominantly from the layer formed by the analyte-binding molecules andbound analyte, rather than from the interface region. One exemplarymaterial forming the thin-film layer is SiO₂, e.g., a high-quality glasshaving an index of refraction of about 1.4-1.5. The thin-film layer canalso be formed of a transparent polymer as the monolithic substrate,such as polystyrene or polyethylene, having an index of refractionpreferably in the 1.3-1.8 range.

The thickness of the biomolecular (analyte-binding molecular) layer 21is designed to optimize the overall sensitivity based on specifichardware and optical components. Conventional immobilization chemistriesare used in chemically, e.g., covalently, attaching a layer ofanalyte-binding molecules to the lower surface of the optical element.For example, a variety of bifunctional reagents containing a siloxanegroup for chemical attachment to SiO₂, and an hydroxyl, amine, carboxylor other reaction group for attachment of biological molecules, such asproteins (e.g., antigens, antibodies), or nucleic acids. It is also wellknown to etch or otherwise treat glass a glass surface to increase thedensity of hydroxyl groups by which analyte-binding molecules can bebound. When the thin-film layer is formed of a polymer, such aspolystyrene, a variety of methods are available for exposing availablechemically-active surface groups, such as amine, hydroxyl, and carboxylgroups.

The analyte-binding layer is preferably formed under conditions in whichthe distal surface of the optical element is densely coated, so thatbinding of analyte molecules to the layer forces a change in thethickness of the layer, rather than filling in the layer. Theanalyte-binding layer can be either a monolayer or a multi-layer matrix.

The measurement of the presence, concentration, and/or binding rate ofanalyte to the optical assembly is performed by the interference ofreflected light beams from the two reflecting surfaces in the opticalassembly. Specifically, as analyte molecules attach to or detach fromthe surface, the average thickness of the first reflecting layer changesaccordingly. Because the thickness of all other layers remains the same,the interference wave formed by the light waves reflected from the twosurfaces is phase shifted in accordance with the thickness change due tothe analyte binding.

The use of a monolithic substrate material instead of an optic fiber inthe optical assembly has several advantages. In a preferred embodiment,the refractive index of the monolithic substrate is higher than therefractive index of the transparent material of the thin-film layer.Because the monolithic substrate is a single solid material, therefore,it is easy to select a material having higher refractive index than thatof the thin-film layer. On the contrary, an optic fiber is typically acircular cross-section dielectric waveguide consisting of a dielectricmaterial (a core material) surrounded by another dielectric materialwith a lower refractive index (cladding); therefore, it is difficult tomanipulate its refractive index. In the prior art (U.S. Pat. No.7,394,547), the refractive indexes of the optical sensor fiber and theinterference layer are essentially the same having values ofapproximately 1.46. Hence, the prior art requires an extra coating of ahigh refractive material on top of the interference layer in order toproperly reflect the incident light.

In operation, incident light signal 25 is emitted from the light source11 and is transported through the waveguide 13 wherein the incidentlight signal 25 is coupled through the coupling medium 16 to themonolithic substrate 17 and subsequently coupled to the thin film layerand the biomolecular layer 21. Within the optical assembly 14, light isreflected at the second reflecting surface 23 resulting in a reflectedlight signal 26. Additionally, light is reflected at the firstreflecting surface 28 resulting in the reflected light signal 27. Beforeanalyte binding, the first reflecting surface is a surface between alayer of biomolecules (analyte binding molecules) 21 and the samplesolution. After analyte binding, the first reflecting surface becomesthe surface between a layer analyte molecules and the sample solution.

The two light signals reflected from boundaries between first and secondrefracting surfaces generate a spectral interference pattern, as shownin FIG. 2 a. When biomolecules bind to analyte molecules on theperipheral surface of the thin-film layer (interference layer), thesecond reflection signal's equivalent optical path extends. As a result,the spectral interference pattern shifts from T0 to T1 as shown in FIG.2 b. By measuring the pattern's phase shift continuously in real-time, akinetic binding curve can be measured as the amount of shift vs. thetime. The association rate of an analyte to a capture moleculeimmobilized on the surface can be used to calculate the analyte'sconcentration. Hence, the measurement of this phase shift is thedetection principle of a thin-film interferometer.

Referring to FIG. 2 a, the performance of the bio-sensor interferometeris improved as the AC component is maximized and the DC offset isminimized. To achieve these objectives, one must increase the couplingefficiency for the incident light signal 25 from the light source 11 tothe sensing surface 24 and the coupling efficiency for the reflectedlight signals 26 and 27 from the first and second refracting surfaces tothe spectrometer 12. Concurrently, the reflection from the otherinterfaces should be reduced as much as possible.

In one embodiment, the present invention comprising a coupling hub isillustrated in FIG. 3, where a probe 32 is inserted into the centerbores of the molded plastic 31 and results a structure 33. The probeswere then inserted into the center bores of the molded plastic hub. FIG.4 shows a simplified illustration of the bio-sensor based on thin filminterferometer. The bio-sensor comprises light source 11, spectrometer12, waveguide 13, ferrule 41, coupling hub 31, probe 42, and sensingsurface 24. The tip of the probe and the sensing surface 24 are dippedinto a coating solution containing analyte-binding molecules.

This invention is directed to an assembly for use in detecting ananalyte in a sample based on thin-film spectral interference. Theassembly comprises: (a) a waveguide connector containing a waveguide,wherein the waveguide transports a light signal from a light source tothe assembly, and the waveguide transports reflected light signals fromthe assembly to a detector; (b) a monolithic substrate having a couplingside and a sensing side, the coupling side is coupled to the waveguideconnector with the waveguide by a coupling hub; (c) a coupling mediumlocated between the waveguide connector and the monolithic substrate sothe waveguide optically couples with the monolithic substrate withoutany gap; and (d) a thin-film layer directly bonded to the sensing sideof the monolithic substrate, wherein the thin film layer comprises atransparent material, a first reflecting surface comprising a layer ofanalyte binding molecules, and a second reflecting surface between thethin film layer and the monolithic substrate; whereby a spectralinterference between light reflected into the waveguide from theassembly varies as analyte molecules in the sample bind to the analytebinding molecules.

In one embodiment, the coupling hub is inserted into the waveguideconnector. In another embodiment, the waveguide connector is a ferruleand the ferrule is inserted into the coupling hub.

In one embodiment, the coupling hub comprises the coupling medium. Inanother embodiment, the waveguide connector is a ferrule and thecoupling medium is between the ferrule and the bottom of the couplinghub.

In FIG. 5, bio-sensor 50 illustrates the present invention implementedwith a coupling hub 51. Bio-sensor 50 is an assembly for use indetecting an analyte in a sample based on thin-film spectralinterference. Bio-sensor 50 comprises a ferrule 55 containing awaveguide 13, wherein the waveguide 13 transports the incident lightsignals from the light source 11 to the coupling hub 51 and transportsreflected light signals from the coupling hub 51 to the spectrometer 12(detector). Bio-sensor 50 further comprises a probe 42, which is coupledto the ferrule 55 with the waveguide 31 by a coupling hub 51, andferrule 55 is inserted into the coupling hub 51. Within the coupling hub51, a coupling medium 53 is located between the ferrule 55 and the probe52 so the end of the waveguide 31 directly contacts the monolithicsubstrate of the probe 42 without any gap, such as an air gap.

FIG. 6 illustrates one coupling hub embodiment of FIG. 5 with a couplinghub assembly 60. Coupling hub 51 has a coupling medium 16 pre-installedinside and at the bottom of coupling hub 51, as indicated in FIG. 6 a.Probe 42 contacts the coupling medium 16 at the bottom of the hub. Asillustrated in FIG. 6 b, a ferrule 55 with a fiber bundle (waveguide 13)is inserted into coupling hub 51. As illustrated in FIG. 6 c, thecoupling medium 16 connects probe 42 and the instrument fiber bundle(waveguide 13) without a gap. Hence, the coupling medium 16 is locatedbetween the ferrule 55 and the bottom of the coupling hub 51.

FIG. 7 illustrates a second coupling hub embodiment of FIG. 5 withcoupling hub assembly 70 having a droplet of coupling medium formed onthe tip of the ferrule. FIG. 7 a shows a ferrule 55 with fiber bundle(waveguide 13), which is dipped into a reservoir 65 of coupling mediumof index matching gel or liquid (FIG. 7 b). In FIG. 7 c, the ferrule 55with fiber bundle (waveguide 13) is removed from the reservoir 65resulting in a droplet of coupling medium 16 formed on the tip of theferrule 55. In FIG. 7 d, the resulting ferrule 55 with fiber bundle(waveguide 13) is inserted into coupling hub 51. The result is that thedrop of the coupling medium 16 connects probe 42 and the instrumentfiber bundle (waveguide 13) with zero gap coupling, as illustrated inFIG. 7 e. Hence, the drop of coupling medium 16 is located between theferrule 55 and the bottom of the coupling hub 51.

FIG. 8 illustrates a third coupling hub embodiment of FIG. 5 withcoupling hub assembly 80 having a coupling medium 81 pre-installed onferrule 55, as indicated in FIG. 8 a. In FIG. 8 b, the ferrule 55 withcoupling medium 81 and with fiber bundle (waveguide 13) is inserted intocoupling hub 51. The result is that coupling medium 81 connects probe 42and the instrument fiber bundle (waveguide 13) without any gap, asillustrated in FIG. 8 c. Hence, the coupling medium 81 is locatedbetween the ferrule 55 and the bottom of the coupling hub 51.

FIG. 9 illustrates a fourth coupling hub embodiment of FIG. 5 withcoupling hub assembly 90 comprising a coupling medium such as a polymermaterial. As shown in FIG. 9 a, coupling hub 92 comprises a couplingmedium. In FIG. 9 b, a ferrule 55 with fiber bundle (waveguide 13) isinserted into coupling hub 51. The result is that ferrule 55 contactsthe bottom of coupling hub 92 and the probe 42 contacts the oppositeside of the coupling hub 92 resulting in zero gaps coupling between theferrule 55 and probe 42.

FIG. 10 a-10 c show another coupling hub embodiment with coupling hubassembly 100. Assembly 100 comprises waveguide connector 102 and theremovable probe 101. FIG. 10 a is a perspective view showing thewaveguide connector 102 and the removable probe 101 unattached to eachother. The wave guide connector 102 has a hole 114 in the top where thewaveguide (e.g. fiber) is inserted. FIG. 10 b is a cross-section view ofthe waveguide connector 102 and the removable probe 101 unattached toeach other. FIG. 10 c shows the removable probe 101 inserted into thewaveguide connector 102.

Referring first to FIG. 10 a, the removable probe 101 consists of amonolithic substrate 104, which is coatd with a thin-film layer, and acylindrical shaped coupling hub 103, which has a hole 105 in the center.The monolithic substrate 104 is deposited with a thin-film coating suchas a SiO₂ layer and analyte binding molecules. The monolithic substrate104 is in a rod shape with its length at least 5 times greater than thediameter. As shown in FIG. 10 b, the removal probe has a sensing surface111 for detecting analyte and a coupling surface 112. In one design, thehub 103 is made from transparent plastics. The hole 105 in the hub 103is not a through hole with one end having a layer 108 of the sametransparent plastic material as the hub 103. Layer 108 is the top ofcylindrical shaped hub 103. The substrate 104 is fixedly attached to thehub 103 by inserting the substrate's coupling surface against the insidewall of coupling surface 112 of the layer 108.

This layer 108 serves as an optical coupling medium between thesubstrate's coupling surface 112 and the waveguide 113 that is installedinside the waveguide connector 102. The waveguide connector shown herehas flexible gripping arms 107 to engage the hub 103 and maintain enoughfictional force to hold the substrate 104 in place relative to thewaveguide 113. The inside of the waveguide connector 102 has a flatengagement surface 109 that is flushed with the waveguide's surface. Thehub 103 has a flat top surface 110. When engaged, the top surface 110 ispushed against the engagement surface 109 so that the gap between thetwo surfaces are completely closed. The layer 108 as a coupling mediumis sandwiched between the coupling surface 112 of the substrate 104 andthe waveguide 113. With a coupling medium having the refractive indexgreater than the air, preferably between the waveguide 113 and thesubstrate 104, will enhance the coupling efficiency, minimize theundesirable reflections back to the spectrometer, and reduce thesensitivity of the lateral misalignment between the substrate 104 andthe waveguide 113.

FIG. 10 c illustrates the engagement of the waveguide connector 102 andthe removable probe 101.

FIG. 11 a-11 c show details of another coupling hub embodiment withcoupling hub assembly 110. Assembly 100 comprises of waveguide connector202 and the removable probe 201. FIG. 11 a is a perspective view showingthe waveguide connector 202 and the removable probe 201 unattached toeach other. FIG. 11 b is a cross-section view of the waveguide connector202 and the removable probe 201 unattached to each other. The wave guideconnector 202 has a hole 114 in the top where the waveguide (e.g. fiber)is inserted. FIG. 11 c shows the removable probe 201 inserted into thewaveguide connector 202.

Referring first to FIG. 11 a, the removable probe 201 consists of amonolithic substrate 104 in a rod shape with its length at least 5 timesgreater than the diameter, and a cylindrical shaped hub 203 that has ahole 205 in the center. As shown in FIG. 11 b, the substrate 104 has asensing surface 111 and a coupling surface 112. The hub 203 is made fromtransparent plastics. The substrate 104 is fixedly attached to the hub203 by inserting the substrate into the hole until the coupling surface112 flush with the hub's top surface 210. The sensing surface 111 isdeposited with a thin-film coating that includes a SiO2 layer andanalyte binding molecule that can be used for detecting analyte.

A coupling medium 213 is installed inside the waveguide connector 202 atthe end of the waveguide 113. The coupling medium is preferred to choosefrom optically transparent, elastic materials or liquid-like materials.PDMS is an example. The waveguide connector 202 shown here has flexiblegripping arms 107 to engage the hub 203 and maintain enough fictionalforce to hold the substrate 104 in place relative to the waveguide 113.

When engaged, the hub's top surface 210 is pushed against the couplingmedium 213 so that the gap between the waveguide 113 and the substrate104 is completely closed. The coupling medium 213 is sandwiched betweenthe coupling surface 112 of the substrate 104 and the waveguide 113.With a coupling medium having the refractive index greater than the air,preferably between the waveguide 113 and the substrate 104, will enhancethe coupling efficiency, minimize the undesirable reflections back tothe spectrometer, and reduce the sensitivity of the lateral misalignmentbetween the substrate 104 and the waveguide 113.

FIG. 11 c illustrates the engagement of the waveguide connector 202 andthe removable probe 201.

As previously noted, a waveguide may be provided by a Y-shaped opticfiber coupler (Y-coupler). An improved embodiment of a Y-shaped opticfiber coupler is illustrated in FIG. 12. Embodiment 100 comprises alight source 11, a spectrometer 12, and a Y-coupler with uneven branches121 coupled to the light source 11 and spectrometer 12. In order toimprove the performance of the bio-sensor, the size of the connection tothe spectrometer 12 (detector) is designed to be larger that the size ofthe connection to the light source 11. As shown in FIG. 12, when aY-shape optic fiber coupler is used, it is preferred to fuse an opticfiber having a smaller diameter with an optic fiber having a largerdiameter. The smaller arm will be used for the illuminating light fromlight source 11 and the larger one for the spectrometer 12. Thisconfiguration will enable more reflecting light go to the spectrometer12, thus improve the overall coupling efficiency.

The present invention is also directed to an assembly for use indetecting an analyte in a sample based on thin-film spectralinterference. The assembly comprises: (a) a waveguide; (b) a monolithicsubstrate having a coupling side and a sensing side, the coupling sideis optically coupled to the waveguide; (c) a thin-film layer directlybonded to the sensing side of the monolithic substrate; wherein thewaveguide transports a light signal from a light source to the assembly,and the waveguide transports reflected light signals from the assemblyto a detector; the thin film layer comprises a transparent material, afirst reflecting surface comprising a layer of analyte bindingmolecules, and a second reflecting surface between the thin film layerand the monolithic substrate; the refractive index of the monolithicsubstrate is higher than the refractive index of the transparentmaterial of the thin-film layer; whereby a spectral interference betweenlight reflected into the waveguide from said first and said secondreflecting surfaces varies as analyte molecules in the sample bind tothe analyte binding molecules. In this assembly, all the terms have thesame characteristics as those recited before.

In one design, the assembly eliminates the gap between the waveguide andthe monolithic substrate by connecting them end to end. Alternatively,the assembly further comprises a coupling medium between the waveguideand the monolithic substrate to fill any gap in between. The refractiveindex of the coupling medium is in general greater than about 1.3,preferably in between refractive indexes of the waveguide and themonolithic substrate.

In yet another design, there is a gap between the waveguide and themonolithic substrate.

The apparatuses described in this application can be used for thefollowing applications: (i) with an anti-species antibody carried on thetip, for screening hybridoma expression lines for cell lines with highantibody expression; (ii) with an antigen carried on the tip, tocharacterize high affinity antibodies against that antigen; (iii) with aprotein carried on the tip, for identifying and characterizing bindingpartners (DNA, RNA, proteins, carbohydrates, organic molecules) for thatprotein; (iv) with a carbohydrate or glycosyl moiety carried on the tip,for identifying and characterizing binding partners (such as, e.g., DNA,RNA, proteins, carbohydrates, organic molecules) for that carbohydrate;(v) with a protein that participates in a multi-protein complex carriedon the tip, for characterizing the binding components and/or kinetics ofcomplex formation; (vi) with a small protein-binding molecule carried onthe tip, for identifying and characterizing protein binders for thatmolecule; (vii) with an antibody carried on the tip, for constructing acalibration curve for the analyte using a set of analytes standards.Using this calibration curve, one can then determine the concentrationof the analyte in unknown solutions (cell culture supernatants,biological samples, process mixtures, etc). (viii) with asingle-stranded nucleic acid, e.g., ssDNA or RNA carried on the tip, foridentifying and molecules that bind specifically to the nucleic acid.

The invention is illustrated further by the following examples that arenot to be construed as limiting the invention in scope to the specificprocedures described in them.

EXAMPLES Example 1 Preparation of Streptavidin Probes

A glass rod (a monolithic substrate), 1 mm diameter and 2 cm in length,had both coupling end and sensing end polished. The sensing end wasfirst coated with a SiO₂ coating layer (a thin-film layer) with athickness of 650 nm using a physical vapor deposition technology, andthen deposited with aminopropylsilane (APS) using a chemical vapordeposition process (Yield Engineering Systems, 1224P) followingmanufacturer's protocol. APS is deposited to enable proteinimmobilization. APS adsorbs protein to the surface of the probe by acombination of hydrophobic and ionic interaction. Protein can also becoupled to the amino group of APS by covalent coupling using acrosslinking reagent. APS is only a monolayer, about 7 nm thick.

The probe (the glass rod coated with SiO₂ and APS) was then insertedinto the center bores of the molded plastic hubs, as shown in FIG. 3.The probe was then locked to the hub by either adhesive or a lockingmechanism.

The probe tip with the sensing end was then immersed in a solution ofstreptavidin (Scripps Labs), 50 μg/ml in phosphate buffered saline pH7.4 (PBS). After allowing the streptavidin to adsorb to the probe for 5minutes, the probe tip was washed in PBS, then immersed in a solution of10% sucrose for 30 seconds followed by drying at 30° C. for one hour andthen stored in a dry condition. Typically, the probe is at least 0.5 mmimmersed.

Example 2 Detection of Protein A Binding to Human IgG

As shown in FIG. 3, each probe was first connected to waveguides in aferrule. The waveguides connect to a halogen light source and aspectrometer so the light will be transported to the probe's sensingsurface and reflected light will be transported to the spectrometer formeasurement. Multiple of such systems can be set up to achieve paralleldetection. The spectrometers measure the reflected light from thesensing ends of the probes and output spectral interference patterns ina real-time function of wavelengths and light intensity at eachwavelength. A typical interference pattern is shown in FIG. 13. A phasechange in the form of left shift of the peaks and valleys represents areduction of the thin-film thickness (disassociation of molecules on thesensing surface); and a right shift means an increase of the thin-filmthickness (association of molecules on the sensing surface). Byconverting the interference pattern to a digital format, a computer isused to determine the amount of thickness change at any time instance. Abinding curve (or association and disassociation curve) can be graphedwith respect to the time. Kinetics of molecular interactions between twodifferent molecules, one in solution and one immobilized on a probe'ssensing surface, can thus be analyzed. In a quantization assay, theconcentration of the analyte molecules in solution can be derived fromthe kinetics: faster binding kinetics implies higher concentration ofanalyte.

An example is demonstrated with two probes measured in parallel. FIG. 14shows the association and disassociation curves of two probessimultaneously.

To start the analysis, the probe's sensing ends were immersed in PBS forabout 20 seconds to hydrate the immobilized streptavidin (Step 1). Thisstep was also used to establish a baseline for the binding curve. Theprobes were then transferred to a biotinylated Protein A sample (PierceChemical) at 50 μg/ml concentration in PBS in Step 2. After about 100seconds there was about a 1 nm shift. Step 3 entailed a brief wash ofthe probe sensing ends in PBS. Step 4 shows the binding to Human IgG(Jackson ImmunoResearch) at 0.1 mg/ml concentration in PBS to theProtein A coated probe sensing ends. In about two minutes, the totalthickness of the thin-film layer (SiO₂ coating) plus biomolecular layerincreased by 6.5 nm (Step 4). To disassociate the IgG/Protein A complex,the probes were transferred to sodium acetate solution at pH 4. In about75 seconds the thin-film thickness decreased by 3 nm (Step 5).

The invention, and the manner and process of making and using it, arenow described in such full, clear, concise and exact terms as to enableany person skilled in the art to which it pertains, to make and use thesame. It is to be understood that the foregoing describes preferredembodiments of the present invention and that modifications may be madetherein without departing from the scope of the present invention as setforth in the claims. To particularly point out and distinctly claim thesubject matter regarded as invention, the following claims conclude thisspecification.

What is claimed is:
 1. An assembly for use in detecting an analyte in asample based on thin-film spectral interference, comprising: a waveguideconnector containing a waveguide; a monolithic substrate having acoupling side and a sensing side, wherein the coupling side is coupledto the waveguide connector by a coupling hub, and the monolithicsubstrate is removable from the waveguide connector; a coupling mediumlocated between the waveguide connector and the monolithic substrate,wherein the refractive index of the monolithic substrate is higher thanthe refractive index of the waveguide, and the refractive index of thecoupling medium is in between the refractive indices of the monolithicsubstrate and the waveguide; and a thin-film layer directly bonded tothe sensing side of the monolithic substrate, wherein the thin filmlayer comprises a transparent material, a first reflecting surfacecomprising a layer of analyte binding molecules, and a second reflectingsurface between the thin film layer and the monolithic substrate,wherein the waveguide is configured to transport a light signal from alight source to the sensing side of the monolithic substrate, and thewaveguide is further configured to transport light signals from thesensing side of the monolithic substrate to a detector; whereby aspectral interference between light reflected into the waveguide fromsaid first and said second reflecting surfaces varies as analytemolecules in the sample bind to the analyte binding molecules.
 2. Theassembly as in claim 1, wherein the refractive index of the monolithicsubstrate is higher than the refractive index of the transparentmaterial of the thin-film layer.
 3. The assembly as in claim 1, whereinthe refractive index of the coupling medium is greater than 1.3.
 4. Theassembly as in claim 1, wherein the monolithic substrate is a sensorfiber.
 5. The assembly as in claim 1, wherein the coupling hub isinserted into the waveguide connector.
 6. The assembly as in claim 1,wherein the coupling hub comprises the coupling medium.
 7. The assemblyas in claim 1, wherein the waveguide connector is a ferrule and theferrule is inserted into the coupling hub.
 8. The assembly as in claim1, wherein the waveguide connector is a ferrule and the coupling mediumis between the ferrule and the bottom of the coupling hub.
 9. Theassembly according to claim 1, wherein the length to width or length todiameter aspect ratio of the monolithic substrate is at least 5 to 1.10. The assembly according to claim 1, wherein the monolithic substrateis made of glass, quartz, or plastic.
 11. The assembly according toclaim 1, wherein the refractive index of the monolithic substrate is atleast 1.55.
 12. The assembly according to claim 1, wherein a Y-shapedfiber optic coupler is coupled from the waveguide to the light sourceand the detector, wherein a branch coupled to the detector has a largerdiameter than a branch coupled to the light source.
 13. An assembly foruse in detecting an analyte in a sample based on thin-film spectralinterference, comprising: a waveguide; a monolithic substrate having acoupling side and a sensing side, wherein the coupling side is opticallycoupled to the waveguide, and the monolithic substrate is removable fromthe waveguide; a thin-film layer directly bonded to the sensing side ofthe monolithic substrate; a coupling medium located between thewaveguide and the monolithic substrate; and the thin film layercomprises a transparent material, a first reflecting surface comprisinga layer of analyte binding molecules, and a second reflecting surfacebetween the thin film layer and the monolithic substrate; wherein therefractive index of the monolithic substrate is higher than therefractive index of the waveguide, and the refractive index of thecoupling medium is in between the refractive indices of the monolithicsubstrate and the waveguide; wherein the coupling medium has a diameterlarger than a diameter of the monolithic substrate; wherein thewaveguide is configured to transport a light signal from a light sourceto the sensing side of the monolithic substrate, and the waveguide isfurther configured to transport light signals from the sensing side ofthe monolithic substrate to a detector; wherein the refractive index ofthe monolithic substrate is higher than the refractive index of thetransparent material of the thin-film layer; and whereby a spectralinterference between light reflected into the waveguide from said firstand said second reflecting surfaces varies as analyte molecules in thesample bind to the analyte binding molecules.
 14. The assembly accordingto claim 13, further comprising a coupling medium between the waveguideand the monolithic substrate, wherein the refractive index of thecoupling medium is greater than 1.3.
 15. The assembly according to claim13, wherein the length to width or length to diameter aspect ratio ofthe monolithic substrate is at least 5 to
 1. 16. The assembly accordingto claim 13, wherein the monolithic substrate is made of glass, quartz,or plastic.
 17. The assembly according to claim 13, wherein therefractive index of the monolithic substrate is at least 1.55.
 18. Theassembly according to claim 13, wherein a Y-shaped fiber optic coupleris coupled from the waveguide to the light source and the detector,wherein a branch coupled to the detector has a larger diameter than abranch coupled to the light source.
 19. The assembly according to claim1, wherein the coupling medium has a diameter larger than a diameter ofthe monolithic substrate.